The Journal of Experimental Biology 207, 2725-2733 2725 Published by The Company of Biologists 2004 doi:10.1242/jeb.01090

Immune response to fleas in a wild desert rodent: effect of parasite species, parasite burden, sex of host and host parasitological experience Irina S. Khokhlova1, Marina Spinu2, Boris R. Krasnov3,* and A. Allan Degen1 1Desert Adaptations and Husbandry, Wyler Department of Dryland Agriculture, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel, 2University of Agricultural Sciences and Veterinary Medicine, Manastur str. 3-5, Cluj-Napoca, Romania and 3Ramon Science Center and Mitrani Department of Desert Ecology, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, PO Box 194, Mizpe Ramon 80600, Israel *Author for correspondence (e-mail: [email protected])

Accepted 18 May 2004

Summary We studied immune responses of the jird Meriones responses to antigens of fleas familiar to their mothers (X. crassus to different flea species belonging to the same c. mycerini and X. ramesis) were significantly higher family. We used jirds maintained in an outdoor enclosure than those to antigen of S. c. pyramidis and (enclosure; N=18) and parasitized by fleas Xenopsylla phytohemagglutinin. The results clearly demonstrated conformis mycerini and Xenopsylla ramesis, and also jirds that (i) cross-reactivity in rodent responses to different born in the laboratory to previously parasitized mothers flea species occurred for enclosure but not for laboratory (laboratory ; N=23). We asked (i) whether cross- jirds and (ii) immune-naïve animals whose mothers were immunity to different fleas occurs, (ii) whether there is a parasitized by fleas had some degree of immunity against sex difference in immune responses to flea parasitism and fleas. The only sex difference in immunological (iii) whether the severity of the immune responses depends parameters was the higher level of circulating immune on parasite load. In the enclosure animals, immune complexes in females than in males. Only phagocytic response to antigen from the unfamiliar flea Synosternus activity was affected by flea burden, decreasing with an cleopatrae pyramidis did not differ from those to antigens increase in flea numbers. from the familiar fleas. In contrast, laboratory rodents demonstrated no difference in the immune response between S. c. pyramidis antigen and either the Key words: rodent, Meriones crassus, flea, immune response, cross- phytohemagglutinin treatment or controls, although their immunity, maternal transfer, sex difference.

Introduction Ectoparasitic are often thought of as crawling or response to ectoparasitic arthropods such as ticks, mites, flying hypodermic needles that suck blood and inject disease- chiggers, fleas, mosquitoes and lice is well documented causing agents. However, saliva of blood-feeding arthropods (Ribeiro, 1987, 1995; Rechav, 1992; Wikel et al., 1996; Wikel contains factors that help them evade host haemostatic and Alarcon-Chaidez, 2001); however, most studies of defenses (Ribeiro, 1995; Wikel, 1996) and also has potent immune responses to ectoparasites have involved livestock or immunogens that influence the immune responses of the laboratory animals; relatively little is known about immune host (e.g. Roehrig et al., 1992). Ectoparasite arthropods responses to ectoparasites in wild animals. downregulate host innate and specific acquired immune Haematophagy evolved independently in different taxa of defenses, inducing host responses that impair their own ability arthropods and, thus, it is commonly accepted that chemical to feed (Rechav et al., 1989). For example, a study of acquired mediators contained in their saliva are different (Ribeiro, 1995; resistance in guinea pigs to tick larvae showed that repeated Jones, 1996). However, salivary anticlotting, antiplatelet and infestation of the host resulted in a sharp reduction in vasodilatory substances can be quite similar within a parasite body mass of engorged larvae (Fielden et al., 1992). This taxon (e.g. genus and family) (Mans et al., 2002; but see resistance is due to stimulation by ectoparasites of host Warburg et al., 1994). This within-taxon similarity can lead to immunoregulatory and effector responses, which involve cross-resistance (= heterospecific resistance) of a host against antigen-presenting cells, T lymphocytes, B lymphocytes, closely related parasites. For example, cross-resistance to antibodies, complement, mast cells, circulating granulocytes closely related ticks has been repeatedly reported (McTier et al., and cytokines (Jones, 1996). The development of an immune 1981; Njau and Nyindo, 1987; but see Rechav et al., 1989). 2726 I. S. Khokhlova and others

Fleas (Siphonaptera) are parasites of higher vertebrates, However, the cost of using the immune defense system is being most abundant and diverse on small mammals. presumably high and there are numerous trade-offs between Mammals vary in their flea species richness. For example, immune defense and other concurrent needs of an organism among 12 rodent species in the Negev desert, the number of (Sheldon and Verhulst, 1996; Schmid-Hempel and Egert, flea species per rodent species ranged between two and eight 2003). Therefore, a relatively low response of a host can be (Krasnov et al., 1997). Host-dependent and habitat-dependent expected both when (i) parasite pressure is low and (ii) the cost fleas were distinguished. For example, Parapulex chephrenis of eliminating parasites is higher than the cost of limiting its parasitized spiny mice (Acomys cahirinus and Acomys pressure to a ‘tolerable’ level (Combes, 2001). Consequently, russatus) almost exclusively, independent of habitat, whereas responses of the host are expected to peak at intermediate Synosternus cleopatrae pyramidis was found only on sand- levels of parasite load and, thus, the curve describing the dwelling rodents, independent of species affinities (Krasnov et relationship between parasite load and host response level is al., 1999). expected to be hump-shaped. To test this prediction we studied are considered to be monophyletic (Traub, 1980; Smit, immune responses in M. crassus parasitized by different 1982; Whiting et al., 1997). Given the common origin and numbers of fleas. opportunistic feeding of many flea species (Marshall, 1981), Furthermore, it is known that parasite circulating antigens, cross-resistance of hosts to different species of fleas can be immunoglobulins, immune cells, cytokines and other cell- expected. This can be especially true for those hosts that related products can be transferred from mammalian females demonstrate high richness of natural flea assemblages and/or during pregnancy and/or lactation to their young (Carlier and those fleas that have a broad range of natural hosts. However, Truyens, 1995). This can induce a long-term modulation of the immune responses against fleas are poorly known (Jones, offspring’s capacity to mount immune responses to subsequent 1996). No datum supporting or rejecting an existence of exposure to parasites (Carlier and Truyens, 1995). Maternal immune cross-reaction of a host against different flea species transfer of immunity has been suggested for a number of is available except the note by Studdert and Arundel (1988) on protozoans and helminthes (e.g. Heckmann et al., 1967; a severe allergic reaction in cats that hunted rabbits infested Shubber et al., 1981; Kristan, 2002). However, we are unaware with the rabbit flea Spillopsyllus cuniculi. The severity of these of any study on maternal transfer of immunity against symptoms indicated that cats had a much higher response to ectoparasites. We did not measure directly the maternal rabbit fleas than to the cat flea Ctenocephalides felis, with transfer of immunity in our experiments in terms of immune which they were normally infested. Furthermore, differential parameters of placental–fetal circulation or milk. Nevertheless, immune responses of rodents to natural and unnatural flea the occurrence of maternal transfer of immunity could be species have been reported (e.g. Vaughan et al., 1989). inferred from the comparison between immune responses of We studied immune responses of Sundevall’s jird Meriones rodents that were previously parasitized by fleas and those of crassus to different flea species belonging to the same family rodents that were never parasitized but whose dams were (Pulicidae) (Xenopsylla conformis mycerini, Xenopsylla parasitized by fleas. ramesis and Synosternus cleopatrae pyramidis). These fleas have different habitat preferences. M. crassus is parasitized either by Xenopsylla species, which co-occur together in some Materials and methods areas or by S. c. pyramidis, which has a distinct geographic Rodents distribution from Xenopsylla species (Krasnov et al., 1999). Meriones crassus Sundevall is a common rodent species We studied rodents that originated from areas inhabited by X. of southern Israel. It occupies a variety of habitats and is c. mycerini and X. ramesis and predicted that if cross-immunity parasitized naturally by several flea species (Krasnov et al., to different fleas occurs, immune responses of a rodent to a flea 1996, 1997; Table·1). We used rodents from our laboratory species that previously parasitized the rodent (familiar flea, colonies. Progenitors of the colonies were captured at the both Xenopsylla) will be similar to responses to other Ramon erosion cirque, Negev Highlands, Israel (30°35′N, (unfamiliar, S. c. pyramidis) flea species. If, however, cross- 34°45′E) in 1996. In this area, M. crassus are permanently immunity does not exist, immune responses to a familiar flea infested with X. c. mycerini and/or X. ramesis (prevalence of species will differ from those to unfamiliar flea species. infestation about 90–100%; Krasnov et al., 1997), but never In addition, we asked whether there is a sex difference in infested with S. c. pyramidis. Animals were maintained either immune responses to flea parasitism. Sex differences in in an outdoor covered circular enclosure (5·m diameter and immunocompetence and susceptibility to parasites were 1.5·m height) or in an animal room. reported for a variety of mammals, with males being generally The enclosure, established in 2000, was built of wire mesh less immunocompetent and more susceptible to parasites than (1·cm×1·cm) and contained 60·cm layer of natural sandy- females (e.g. Olsen and Kovacs, 1996; Poulin, 1996; Schalk gravel substrate, which allowed rodents to burrow. In addition, and Forbes, 1997). 20 wooden nest boxes with dried grass (bedding material) were If a host immune response is an efficient tool to overcome placed in the enclosure. However, jirds clearly preferred to stay macroparasite pressure, an increased response with an increase in burrows rather than in the nest boxes. Millet seed and alfalfa in parasite load can be expected (de Lope et al., 1998). (Medicago sp.) leaves were provided daily ad libitum. The Immune response to fleas in a desert rodent 2727

Table·1. assemblages of M. crassus in different habitats in southern Israel Habitat type Flea assemblages Sand dunes of the northern Negev desert Synosternus cleopatrae pyramidis, Nosopsyllus iranus theodori Sand dunes of the central Negev desert Xenopsylla conformis mycerini, Xenopsylla dipodilli, N. i. theodori Sandy-gravel plains of the central Negev desert X. c. mycerini, X. dipodilli, Coptopsylla africana, N. i. theodori Loess dry riverbeds of the central Negev desert X. c. mycerini, Xenopsylla ramesis, X. dipodilli, C. africana, Stenoponia tripectinata medialis, N. i. theodori Loess valleys of the central Negev desert Xenopsylla ramesis, X. dipodilli, S. t. medialis, N. i. theodori, Rhadinopsylla masculana

Based on data of Krasnov et al. (1997, 1998, 1999).

enclosure population was started with ten rodents (five males enclosure jirds, whereas the occurrence of endoparasites was and five females), two of which were infested with 20 X. c. not monitored. mycerini each and two with 20 X. ramesis each. At the time of experiments, there were 80 animals in the enclosure. Flea Fleas burden in the enclosure was monitored monthly on 20 X. c. mycerini, X. ramesis and S. c. pyramidis belong to the randomly selected individuals during a year prior to subfamily Xenopsyllinae of family Pulicidae, although no experiments. Within flea sampling period, intensity of datum on genetic distances between these fleas or about their infestation differed among individuals (range 2–50 for X. c. common ancestor is available. X. c. mycerini and X. ramesis are mycerini and 2–43 for X. ramesis). However, intensity of common ectoparasites of gerbils and jirds throughout the infestation of the same individual over time was relatively Middle East and occur mainly in sandy-gravel and loess stable (±15% on average). At the time of experiments, both flea habitats. We recorded X. c. mycerini mainly on M. crassus, G. species occurred on all rodents and flea burden averaged 15.1 dasyurus, Gerbillus henleyi, whereas X. ramesis was found on X. c. mycerini and 10.8 X. ramesis, which was 30% higher than M. crassus, G. dasyurus, Psammomys obesus, and Eliomys the average flea burden in the field (Krasnov et al., 1998). melanurus (Krasnov et al., 1997, 1999). S. c. pyramidis is Nonetheless, rodents reproduced successfully and did not lose widely distributed in sand massifs of Israel and is characteristic body mass. The area is characterized by hot, dry summers for sand-dwelling rodents. We recorded this flea on Gerbillus (mean daily air temperature of July is 34°C) and relatively cold andersoni allenbyi, Gerbillus pyramidum, M. crassus and winters (mean daily temperature of January is 12.5°C) with Meriones sacramenti in sand dunes of northern Negev (Krasnov 100·mm of annual rainfall, all occurring in winter. et al., 1999) as well as on Gerbillus nanus and Gerbillus Eight pregnant females from the enclosure were captured, gerbillus in sandy habitats of the Arava valley (B. R. Krasnov freed from fleas (5–20 X. c. mycerini and 6–18 X. ramesis) and and N. V. Burdelova, unpublished observations). It should be transferred to the animal room. At the moment of capture, noted that progenitors of our M. crassus colony originated from females were on the second half of pregnancy (as indicated non-sandy area where S. c. pyramidis was never found. by body mass, body shape and occurrence of blood in Fleas were obtained from our laboratory colonies started in vaginal smears; I. S. Khokhlova, unpublished observations). 1998–2001 from field-collected specimens on M. crassus (X. Rodents were housed individually in plastic cages c. mycerini and X. ramesis) and G. a. allenbyi (S. c. pyramidis) (60·cm×50·cm×40·cm) at 25°C with a photoperiod of 12·h:12·h using rearing procedures similar to those described by Metzger (L:D). They were fed millet seeds and alfalfa ad libitum. Dried and Rust (1997). An individual rodent host was placed in a grass was provided as bedding material. Newly born rodents cage (60·cm×50·cm×40·cm) that contained a steel nest box were transferred to individual cages 90 days post partum. with a screen floor and a pan containing a mixture of sand and These animals were never subjected to flea parasitism. dried bovine blood (nutrient medium for larvae). Gravid Measurements were done when jirds were 5–7 months old. female fleas left the host and deposited eggs in the substrate Immunological studies were done on 18 jirds (8 males and and bedding material in the nest box. Every 2 weeks, all 10 females) from the enclosure (hereafter referred to as substrate and bedding material were collected from the nest enclosure animals) and on 23 never-parasitized jirds (8 box and transferred into an incubator, where flea development males and 15 females) born in the laboratory (hereafter and emergence took place at 25°C and 75% relative humidity. referred to as laboratory animals) from previously The newly emerged fleas were placed on clean animals. parasitized mothers (eight litters, 2–4 pups per litter). All Colonies of fleas were maintained at 25°C and 75% RH with enclosure animals were 6–11 months old. It was unknown, a photoperiod of 12·h:12·h (L:D). however, whether some of these animals were from the same litter. The intensity of infestation of these jirds by X. c. Procedures mycerini and X. ramesis ranged between 4 and 17 and 5 and Whole body extracts of fleas 22, respectively. No other ectoparasites were found on the We prepared whole body extracts from newly emerged fleas 2728 I. S. Khokhlova and others that did not feed after emergence. Fleas were collected and then plate (100·µl per well). Five variants were tested once for each frozen at –20°C. Mean body masses of newly emerged individual animal, namely (1) untreated control culture, (2) individuals X. c. mycerini, X. ramesis and S. c. pyramidis were phytohaemagglutinin-M (PHA) (1·µl per well) treated culture, 0.17, 0.16 and 0.20·mg, respectively. Frozen fleas (100 (3–5) antigens of X. c. mycerini, X. ramesis and S. c. pyramidis individuals) were stirred in a mortar, mixed with a small (2.5·µl per well) treated cultures. The quantities of both PHA volume of phosphate-buffered saline (PBS) and filtered and antigens were established when using the same technique through plane filter paper to remove remnants of chitin. Then, during preliminary studies as being the most effective in vitro the extracts were centrifuged for 10·min at 3410·g (CN-2060 for the tested species. The cultures were incubated for 18·h at microprocessor control centrifuge, Hsiangtai Machinery 37.5°C and 5% CO2. Glucose concentrations were measured Industry Co. Ltd, Taiwan) and the pellets of antigen were in the initial medium and in all variants at the end of the resuspended in PBS and freeze-dried (Department of incubation period, using a standard (100·mg·dl–1) glucose Microbiology and Immunology, Faculty of Health Science, solution, by means of an orto-toluidine colorimetric test. To do Ben-Gurion University of the Negev, Israel). Before use, the this, 12.5·µl of the cultural supernatant were transferred to antigen was diluted with PBS to about 50% of the initial mass 0.5·ml of orto-toluidine reagent, boiled for 8·min, cooled of the processed fleas and sterilized by filtering through 0.2·µm suddenly in cold water and read in a spectrophotometer filters (Schleicher & Schuell, Inc., Dassel, Germany). at 610·nm wavelength (Unico 2100, United Products Instruments, Inc., Dayton, NJ, USA), using the reagent as Blood samples a blank. The transformation index (TI) was calculated Heparinized (50·i.u.·ml–1) blood samples (150·µl) from as follows: TI%=[(MG–SG)/MG]×100, where TI=blast rodents were collected from the infraorbital sinus of a rodent transformation index, MG=glucose concentration in the initial using Pasteur pipettes. We did not anesthesize the animals culture medium and SG=glucose concentration in the sample before sampling because this had a negative effect on both the after incubation. blood (inducing haemolysis) and the recovery duration of the animals (I. S. Khokhlova and M. Spinu, unpublished Circulating immune complex measurements observations). Blood from each animal was sampled once Measurement of the level of circulating immune complexes weekly with each jird sampled either 2, 3 (two animals) or 4 (CIC) allows evaluation of the molecular clearance capacity at (one animal) times. Of this blood, two samples from each a particular moment. Part of the collected blood was allowed to animal were used for measurements (one for leucocyte blast clot for 30·min at 37°C and then centrifuged at 1308·g for transformation test and carbon particle inclusion test and one 10·min. Sera were removed and kept at –20°C until tested. A for white blood cell count and serum preparation). The third 4.2% polyethylene glycol (PEG) solution in borate buffer was and/or the fourth blood samplings were used when previous used as the precipitating agent, while buffer-treated samples samples were occasionally damaged. As far as we discerned, served as controls for borate-induced precipitation. The rodents recovered fully 1–2·min after blood sampling. The reaction was performed in a 96-well-plate to enhance experimental design was found to be suitable and to meet spectophotometrical readings. Volumes of 196.7·µl of borate requirements of the 1994 Law for the Prevention of Cruelty to buffer and PEG solution, respectively, were mixed with 3.3·µl Animals (Experiments on Animals) of the State of Israel by samples of the serum, for each sample, in parallel wells. The Ben-Gurion University Committee for the Ethical Care and samples were allowed to precipitate at room temperature Use Animals in Experiments (License IL-27-9-2003). (22–23°C) for 60·min, then read spectrophotometrically at a wavelength of 450·nm in the test plate (d=0.5·cm) Haematological and immunological tests (multichannel spectrophotometer SUMAL PE2, Karl Zeiss, White blood cell count Jena, Germany). CIC concentrations, expressed in optical Part of the heparinized blood was diluted 1:10 with Türk density units (ODU) were calculated by subtracting the value solution, kept at room temperature for 3·min and then of the control (serum + buffer) from that of the PEG precipitate. leukocytes were counted in a Bürker–Türk chamber, counting the elements in four corner squares. The mean value was Immunoglobulin measurements multiplied by 10 for the dilution degree and 10 for the height Total immunoglobulin, known as opsonins, play an of the diluted blood layer in the chamber. The values were important role in the ‘first line of defense’, that is innate expressed in number of cells·mm–3. immunity, against aggressors. At a pH·7.4, the electric charge and colloidal stability of gamma globulins are lower than Leukocyte blast transformation test those of serum albumins. Thus, concentrations as low as The leukocyte blast transformation test measures the in vitro 24·mg·l–1 of metal salts precipitate the immunoglobulin. A reactivity of mononuclear cells to sensitizing (in vivo volume of 6.6·µl of serum was mixed with 193.4·µl of a encountered) antigens. Cell growth was quantified by means 0.024% barbital buffer zinc sulphate solution and allowed to of the glucose consumption technique. Part of the blood sample precipitate for 30·min at room temperature (22–23°C). Optical (100·µl) was diluted with four times the amount of RPMI 1640. density (ODU) then was read spectrophotometrically The mixture was distributed in five wells of a 96-sterile-well- (λ=475·nm, d=0.5·cm). Immune response to fleas in a desert rodent 2729

Carbon particle inclusion test (phagocytic activity) Tukey’s Honest Significant Difference (HSD) tests were Phagocytic cells engulf inert particles such as carbon due applied for all multiple comparisons. to the defensive capacity of these cells. 50·µl portions of To avoid an inflated Type I error, we applied Bonferroni heparinized blood were mixed with 2·µl of supernatant of India adjustment of alpha. Significance was accepted at the adjusted ink, which were obtained by centrifugation at 1308·g for alpha level of 0.005. Data are presented as means ± S.E.M. 40·min (CN-2060 microprocessor control centrifuge, Hsiangtai Machinery Industry Co. Ltd, Taiwan). 15·µl of the mixture were transferred immediately to 2·ml of saline and the rest was Results incubated for 15·min at 37°C. Another 15·µl sample was Immune responses of rodents differed among flea species transferred to saline and the incubation was continued to (Table·2, Fig.·1); however, there was no effect of rodent sex 30·min, repeating the operation. All tubes containing saline, on within-treatment (control, PHA, antigens from three flea blood and ink were centrifuged at 419·g and the supernatants species) transformation index (Table·2). Also, responses of were read spectrophotometrically (λ=535·nm, d=1·cm). There enclosure rodents were significantly higher than those of was a decrease in absorbance with time as carbon was laboratory rodents. This explains the significance of the phagocytized. Phagocytic activity index was calculated as the interaction term of Treatment × Place of birth. difference between the natural logarithms of the optical Rodents from the enclosure and laboratory demonstrated densities of the phagocytosis at 0–15·min and 15–30·min similar low levels of spontaneous glucose consumption divided by time (15·min). (Tukey’s HSD test, P=0.9; see Table·2 for degrees of freedom). The transformation index under phytohemagglutinin treatment Data analysis was significantly higher in enclosure jirds than in controls All dependent variables did not deviate significantly from (Tukey’s HSD test, P<0.0001), but laboratory jirds did not normality (Shapiro–Wilk’s tests, W=0.96–0.98, P>0.3) and their differ from controls (Tukey’s HSD test, P=0.9). Responses to variances were homogenous (Levene’s tests, F3,37=0.001–2.46, antigens from X. c. mycerini and X. ramesis were higher than P>0.1). Therefore, parametric statistics were applied. Because those to phytohemagglutinin in both enclosure and laboratory some laboratory animals (as well as presumably some of rodents (Tukey’s HSD tests, P<0.001), although no difference enclosure animals) were offspring of the same mother, we had to between responses to antigens of these two fleas was found in correctly account for within-litter non-independence of animals. either in enclosure or in laboratory rodents (Tukey’s HSD tests, To do this, we performed analysis of variance (ANOVA) for each P=0.8 and P=0.9, respectively). In addition, in the enclosure dependent variable with identification number of a litter as an jirds, the transformation index with S. c. pyramidis antigen did independent factor. These analyses were done for laboratory not differ significantly from those with antigens from the two animals only because the respective data for enclosure animals other flea species (Tukey’s HSD tests, P=0.08 and P=0.2, were not available. No between-litter difference was found in any respectively) (Fig.·1). In contrast, laboratory rodents of the parameters (F8,14=0.25–1.94, P>0.3). In other words, no demonstrated no difference in transformation index between S. difference in maternal effect on any parameter was found among c. pyramidis antigen and either the phytohemagglutinin mothers of laboratory animals. We therefore assumed that the treatment or controls (Tukey’s HSD tests, P=0.8 and P=0.9, same was true for enclosure animals, although we did not know respectively). Finally, transformation indices with any antigen sibling/non-sibling relationships among these jirds and, were significantly higher in the enclosure than in the laboratory consequently, were unable to perform the respective analysis. rodents (Fig.·1, Tukey’s HSD tests, P=0.4–0.9). Because the same parameter (transformation index) to four The concentration of immunoglobulins was similar in the antigens (PHA and antigens of three flea species) as well as the spontaneous transformation index were measured in each individual, we analyzed the effects of flea species, sex and Table·2. Summary of the repeated-measures ANOVA of the place of birth (enclosure versus laboratory) of a rodent on results of transformation test in dependence on rodent place immune responses using repeated-measures ANOVA with the of birth, rodent sex and treatment (control, PHA, antigens transformation index being a within-subjects factor and place from three flea species) of birth and sex being between-groups factors. The effects of Sum of sex and place of birth on non-specific immune responses were Effect squares d.f. FP analyzed using two-way ANOVAs with immunological Place of birth 0.14 1 6.09 0.018 parameters as dependent variables and flea burden as a Sex 0.073 1 3.14 0.084 continuous predictor (to remove the possible confounding Sex × Place of birth 0.0007 1 0.03 0.867 effect of the difference in flea burden in the enclosure animals). Error 0.86 37 The effect of flea burden on the severity of immune Treatment 0.34 4 64.27 >0.0001 responses was analyzed by simple linear regression as well as Treatment × Place of birth 0.045 4 8.55 >0.0001 2 × linear regression using the equation y=b0+b1x–b2x . The Treatment Sex 0.008 4 1.61 0.17 × × significance of the quadratic term would signify the occurrence Treatment Place of birth Sex 0.027 4 5.01 >0.001 of the peak of immune response at intermediate flea burden. Error 0.198 148 2730 I. S. Khokhlova and others

80 Fig.·1. Transformation index (mean ± S.E.M.) of leucocytes of M. crassus (N=41) in presence of different antigens. PHA, phytohaemagglutinin; Xcm, Xr, Scp, whole body extracts of X. c. mycerini, X. ramesis and 75 S. c. pyramidis, respectively.

70 (Table·3); this activity decreased significantly with an increase in flea burden (Fig.·2). 65

Transformation index (%) Discussion 60 The results of the leukocyte blast transformation tests clearly demonstrated that (i) cross-reactivity in rodent responses to different flea species occurred for the enclosure but not for 55 Control PHA Xcm Xr Scp Control PHA Xcm Xr Scp the laboratory jirds and (ii) immune-naïve animals Enclosure Laboratory whose mothers were parasitized by fleas had some degree of immunity against fleas. The former was confirmed by immune responses of the enclosure enclosure and laboratory animals as well as in males animals to flea antigens in that they did not differ between and females (F1,37=0.10, P=0.7 and F1,37=1.99, P=0.2, familiar (X. c. mycerini and X. ramesis) and non-familiar (S. c. respectively). The level of circulating immune complexes also pyramidis) fleas. The latter was supported by immune did not differ between the enclosure and laboratory rodents responses of naïve rodents born from parasitized mothers to (F1,37=0.46, P=0.5) but was significantly higher in females antigens of X. c. mycerini and X. ramesis. than males (adjusted least squares means 0.05±0.006 versus Cross-immunity (=cross-resistance) was reported for a 0.02±0.009 ODU, respectively, F1,37=8.77, P=0.005). number of parasite taxa such as protozoans (Leemans et al., The number of white blood cells was significantly lower in 1999), gastrointestinal parasites (Smith and Archibal, 1969), the enclosure than in the laboratory animals (adjusted least blackflies (Cross et al., 1993), ticks (Kaiser et al., 1982; squares means 9663.4±844.7 versus 13931.0±897.9·mm–3, Rechav, 1992). Moreover, cross-immunity between distantly respectively, F1,37=10.16, P=0.002), but there was no sex related parasite taxa was also reported. Rabbits infested with difference in this parameter (F1,37=1.88, P=0.2). Phagocytic mites Prosoptes cuniculi produced antibodies reactive with activity was significantly higher in the enclosure animals than both mite and tick extracts, whereas mite-free rabbits did not in the laboratory animals (F1,37=10.09, P=0.002), but did not (den Hollander and Allen, 1986). However, some studies did differ between sexes (F1,37=5.83, P=0.02). not find cross-immunity against different parasite species (e.g. Only phagocytic activity was affected by flea burden Rechav et al., 1989). Apparently, the occurrence of cross-

Table·3. Summary of regressions of haematological and immunological parameters against flea burden Equation 2 y=b0+b1x, d.f.=1,16 y=b0+b1x–b2x , d.f.=2,15 Parameter r2 FPr2 FP Circulating immune complexes 0.02 0.2 0.6 0.28 3.5 0.07 Concentration of total immunoglobulins 0.04 0.8 0.4 0.07 0.4 0.7 White blood cell count 0.05 0.8 0.4 0.07 0.4 0.7 Phagocytic activity 0.53 13.7 0.003 0.45 6.6 0.01 Spontaneous leukocyte blast transformation 0.04 0.4 0.6 0.23 1.3 0.4 Leukocyte blast transformation in the presence of: PHA 0.03 0.4 0.6 0.19 2.0 0.2 X. c. mycernini antigen 0.01 0.01 0.09 0.50 3.4 0.09 X. ramesis antigen 0.05 0.4 0.5 0.40 4.1 0.06 S. c. pyramidis antigen 0.01 0.7 0.8 0.45 2.9 0.1

PHA, phytohaemagglutinin. Immune response to fleas in a desert rodent 2731

11 mother (if any, see below) was specific, aimed against certain 10 • flea species, but could not protect against other, albeit closely related species. 9 Significant immune responses to X. c. mycerini and X. • 8 • ramesis in rodents born in the laboratory suggest a possibility • • 7 • that they received some protection against these fleas from • • their mothers. The occurrence of maternal transfer of immunity 6 • • could be inferred from the difference in immune responses • 5 between the enclosure and laboratory animals, although our

Phagocytic index 4 • study lacks the direct measurements of the maternal transfer of immunity. Maternal transfer of immunity against parasites was 3 reported (Carlier and Truyens, 1995), although the protective 2 • effect of maternal antibodies transfer to offspring is limited • 1 (e.g. Knopf and Coghlan, 1989). Indeed, the responses to 0 2 4 6 8 1012141618202224 antigens of both Xenopsylla species in the laboratory jirds were Number of fleas lower than those in the enclosure animals, suggesting that the Fig.·2. Relationship between phagocytic activity in M. crassus (N=18) protective level of maternal immunity was probably lower than in dependence on flea burden. the acquired immunity against the same flea species. However, the relatively short lifespan of the immune cells that could be supposedly transferred from mothers to offspring suggests immunity depends on both parasite and host taxon. For higher probability of finding them in juvenile individuals rather example, McTier et al. (1981) showed that in guinea pigs, than in young adults, as was the case in our study. cross-immunity occurred between two ticks of Dermacentor An alternative (to maternal transfer of immunity) genus but not between ticks belonging to Dermacentor and explanation for our findings of the occurrence of the immune Amblyomma genera. Rabbits demonstrated cross-resistance response to antigens of two Xenopsylla fleas in the laboratory between two ticks of Hyalomma genus (Kumar and Kumar, jirds could be that the long association between M. crassus and 1996), but not between Rhipicephalus and Ixodes genera X. c. mycerini and X. ramesis in the past induced host (Rechav et al., 1989). Furthermore, cross-immunity between genotypical changes via selection. In particular, these changes two Rhipicephalus species was reported for rabbits (Njau and could affect the major histocompatibility complex, which is the Nyindo, 1987), whereas no cross-immunity between these two region of the genome that controls the immune response ticks was found in guinea pigs (Rechav et al., 1989). (Gruen and Weissman, 1997). The occurrence of cross-immunity can be explained by close Overall low responses in the laboratory jirds (including homology of saliva proteins in closely related ectoparasites relatively low phagocytic activity – see below) as well as the (e.g. Mans et al., 2002). However, this is not always the case lack of their response to phytohemagglutinin can be explained (e.g. Warburg et al., 1994). Furthermore, a variety of cross- by the lack of challenges in the controlled laboratory immunity patterns was also demonstrated with the ‘concealed’ environment and, thus, a ‘lazy’ reactivity of immune cells, ectoparasite antigens (Willadsen and Kemp, 1988; Willadsen mainly lymphocytes and monocytes. In contrast, the enclosure et al., 1993). Bm86 antigen from the tick Boophilus microplus animals probably had more intense change in their contained in the commercial anti-tick vaccine has close polymorphonuclear cells because they were likely to be homologues in both Hyalomma anatolicum (de Vos et al., exposed to permanent microbiological challenges. Therefore, 2001) and Rhipicephalus appendiculatus (Willadsen, 2001). their immune cells were also more active, that is functionally However, a significant level of cross-protection was found ‘faster’, than those of the animals from the laboratory. between the B. microplus vaccine and H. anatolicum (F. Moreover, even though enclosure jirds were permanently Jongejan, unpublished; cited by Willadsen, 2001) but not parasitized, there still may be some effects of maternal transfer between this vaccine and R. appendiculatus (de Vos et al., of immune factors that facilitate their ability to mount immune 2001). It is apparent that some still unknown factors determine response. Further manipulative experiments are needed to test cross-immunity patterns in different host-parasite systems. In this hypothesis (e.g. comparison of acquired immunity against addition, different components of the immune system vary fleas between rodents born from parasitized and not-parasitized interspecifically, and the determinants of such variation are mothers). unknown. The only sex difference in immunological parameters was Our results demonstrate the occurrence of cross-immunity the higher level of circulating immune complexes in females against different flea species in the enclosure but not in the than in males, indicating higher synthesis of antibodies and laboratory jirds. Indeed, the transformation index of clearance of the antigen through complexation in females than leukocytes of laboratory animals in presence of S. c. pyramidis in males. This supports the hypothesis of sexual dimorphism antigen did not differ from the spontaneous transformation in immunocompetence and reduced humoral and cell-mediated index. This suggests that the immunity transferred by the immunity in males (Billingham, 1986; Schuurs and Verheul, 2732 I. S. Khokhlova and others

1990; Zuk and McKean, 1996). Lower immunocompetence in comments. This study was supported by the Israel Science males has been explained by immunosuppressive function of Foundation (Grant no. 663/01-17.3 to B.R.K. and I.S.K.). M.S. androgens (Folstad and Karter, 1992) and has been reported in received financial support from the Access to Research several vertebrate taxa (Hughes and Randolph, 2001; Uller Infrastructure (ARI) program of the EU via the Blaustein and Olson, 2003). Moreover, sexual difference in Center for Scientific Cooperation of the Jacob Blaustein immunocompetence is supported mainly for rather Institute for Desert Research, Ben-Gurion University of the than helminth parasites (Schalk and Forbes, 1997). For Negev. The experiments comply with the laws of the State of example, testosterone treatments reduced both innate and Israel. This is publication no. 160 of the Ramon Science Center acquired resistance of rodents Clethrionomys glareolus, and and no. 412 of the Mitrani Department of Desert Ecology. Apodemus sylvaticus to the feeding of the tick Ixodes ricinus (Hughes and Randolph, 2001). We found sex differences in the humoral component of immunity only, which suggests that References androgens suppressed some but not all components of the Billingham, R. E. (1986). Immunological advantages and disadvantages of immune defense system. Indeed, studying immune responses being a female. In Reproductive Immunology (ed. D. A. Clarke and B. A. in Microtus ochrogaster and Microtus pennsylvanicus, Klein Croy), pp. 1-9. NY: Elsevier Science Publications. Carlier, Y. and Truyens, C. (1995). Influence of maternal infection on and Nelson (1998a) did not observe sex differences in the offspring resistance towards parasites. Parasitol. Today 11, 94-99. proliferative response of splenocytes to concanavalin (cell- Combes, C. (2001). Parasitism. The Ecology and Evolution of Intimate mediated immunity), but found differences in humoral Interactions. Chicago: University of Chicago Press. Cross, M. L., Cupp, E. W., Ramberg, F. B. and Enriquez, F. J. (1993). immunity responses (Klein and Nelson, 1998b). Antibody responses of BALB/c mice to salivary antigens of hematophagous Our prediction about the peak level of host immune response black fleas (Diptera: Simuliidae). J. Med. Entomol. 30, 725-734. at the intermediate parasite burden was not supported. The only de Lope, F., Moller, A. P. and de la Cruz, C. (1998). Parasitism, immune response and reproductive success in the house martin Delichon urbica. parameter that correlated with the number of parasites was Oecologia 114, 188-193. phagocytic activity that decreased with an increase of flea de Vos, S., Zeinstra, L., Taoufik, O., Willadsen, P. and Jongejan, F. (2001). burden. Apparently, even the weak attack of a parasite Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species. Exp. Appl. Acarol. 25, 245-261. triggered the immune system. However, this system could not den Hollander, N. and Allen, J. R. (1986). Cross-reactive antigens between overcome the attack by large number of fleas, perhaps due to a tick Dermacentor variabilis (Acari: Ixodidae) and a mite Prosoptes their additive immunosuppression effect and the cost of the cuniculi (Acari: Psoroptidae). J. Med. Entomol. 23, 44-50. Eiseman, C. H. and Binnengton, K. C. (1994). The peritrophic membrane immune system (Schmid-Hempel and Egert, 2003). – its formation, structure, chemical-composition and permeability in Finally, the difference between parasitized (enclosure) and relation to vaccination against ectoparasitic arthropods. Int. J. Parasitol. non-parasitized (laboratory) animals in the number of white 24, 15-26. Fielden L. J., Rechav, Y. and Bryson, N. R. (1992). Acquired immunity to blood cells, leukocyte blast transformation index and larvae of Amblyomma marmoreum and A. hebraeum by tortoises, guinea- phagocytic activity, but not in the concentration of pigs and guinea-fowl. Med. Vet. Entomol. 6, 251-254. immunoglobulins and circulation immune complexes, Folstad, I. and Karter, A. J. (1992). Parasites, bright males and the immunocompetence handicap. Amer. Nat. 139, 603-622. suggested that the immune response to flea parasitism was Greene, W. K., Carnegie, R. L., Shaw, S. E., Thompson, R. C. A. and linked mainly to cell-mediated immunity. The same has been Penhale, W. J. (1993). Characterization of allergens of the cat flea, shown to be true for the immunity against ticks (e.g. Rubaire- Ctenocephalides felis: detection and frequency of IgE antibodies in canine sera. Parasite Immunol. 15, 69-74. Akiki and Mutinga, 1980). For example, there was no Gruen, J. R. and Weissman, S. M. (1997). Evolving views of the major correlation between rabbit serum antibodies to soluble histocompatibility complex. Blood 90, 4252-4265 antigens from tick salivary gland extracts and protective Heckmann, R., Gansen, B. and Hom, M. (1967). Maternal transfer of immunity to rat coccidiosis – Eimeria nieschulzi (Dieben – 1924). J. immunity (Heller-Haupt et al., 1996). Yet production of anti- Protozool. S14, 35. flea antibodies and transfer of resistance to fleas with immune Heath, A. W., Arfsten, A., Yamanaka, M., Dryden, M. W. and Dale, B. serum were also reported (Greene et al., 1993; Heath et al., (1994). Vaccination against the cat flea Ctenocephalides felis felis. Parasite Immunol. 16, 187-191. 1994; Jones, 1996). Thus, humoral factors can also have a role Heller-Haupt, A., Kagaruki, L. K. and Varma, M. G. R. (1996). Resistance in host resistance to fleas. Furthermore, the digestion in fleas and cross-resistance in rabbits to adults of three species of African ticks is intracellular and they lack a peritrophic membrane (Acari: Ixodidae). Exp. Appl. Acarol. 20, 155-165. Hughes, V. L. and Randolph, S. E. (2001). Testosterone depresses innate (Vaschenok, 1988), which lines the gut of many arthropods. and acquired resistance to ticks in natural rodent hosts: a force for It separates ingested food from the gut epithelium and, thus, aggregated distributions of parasites. J. Parasitol. 87, 49-54. may restrict penetration of ingested immune effector Jones, C. J. (1996). Immune responses to fleas, bugs and sucking lice. In The Immunology of Host-Ectoparasitic Arthropod Relationships (ed. S. K. components (Eiseman and Binnengton, 1994). These two Wikel), pp. 150-174. Wallingford, UK: CAB International. factors can increase the detrimental effect of soluble antigens Kaiser, M. N., Sutherts, R. W. and Bourne, A. S. (1982). Relationship on fleas. between ticks and Zebu cattle in Southern Uganda. Trop. Anim. Health Product. 14, 63-74. Klein, S. L. and Nelson, R. J. (1998a). Sex and species differences in cell- We thank staff of the Department of Microbiology and mediated immune responses in voles. Can. J. Zool. 76, 1394-1398. Immunology, Faculty of Health Science, Ben-Gurion Klein, S. L. and Nelson, R. J. (1998b). Adaptive immune responses are linked to the mating system of arvicoline rodents. Amer. Nat. 151, 59-67. University of the Negev for their help in freeze-drying of Knopf, P. M. and Coghlan, R. L. (1989). Maternal transfer of resistance to antigen pellets. We thank two anonymous referees for helpful Schistosoma mansoni. J. Parasitol. 75, 398-404. Immune response to fleas in a desert rodent 2733

Krasnov, B. R., Shenbrot, G. I., Khokhlova, I. S. and Ivanitskaya, E. Y. associated with rabbit resistance to Rhipicephalus appendiculatus (1996). Spatial structure of rodent community in the Ramon erosion cirque, (Neumann) infestations. Bull. Anim. Health Prod. Africa 28, 49-59. Negev highlands (Israel). J. Arid Environ. 32, 319-327. Schalk, G. and Forbes, M. R. (1997). Male biases in parasitism of mammals: Krasnov, B. R., Shenbrot, G. I., Medvedev, S. G., Vatshenok, V. S. and effects of study type, host age, and parasite taxon. Oikos 78, 67-74. Khokhlova, I. S. (1997). Host-habitat relations as an important determinant Schmid-Hempel, P. and Egert, D. (2003). On the evolutionary ecology of of flea assemblages (Siphonaptera) on rodents in the Negev Desert. specific immune defence. Trends Ecol. Evol. 18, 27-32. Parasitology 114, 159-173. Schuurs, A. H. M. N. and Verheul, H. A. M. (1990). Effects of gender and Krasnov, B. R., Shenbrot, G. I., Medvedev, S. G., Khokhlova, I. S. and sex steroids on the immune response. J. Steroid Biochem. 35, 157-172. Vatschenok, V. S. (1998). Habitat-dependence of a parasite-host Sheldon, B. C. and Verhulst, S. (1996). Ecological immunology: costly relationship: flea assemblages in two gerbil species of the Negev Desert. J. parasite defences and trade offs in evolutionary ecology. Trends Ecol. Evol. Med. Entomol. 35, 303-313. 11, 317-321. Krasnov, B. R., Hastriter, M., Medvedev, S. G., Shenbrot, G. I., Shubber, A. H., Lloyd, S. and Soulsby, E. J. L. (1981). Infection with Khokhlova, I. S. and Vaschenok, V. S. (1999). Additional records of fleas gastrointestinal helminths – effect of lactation and maternal transfer of (Siphonaptera) on wild rodents in the southern part of Israel. Israel J. Zool. immunity. Parasitol. Res. 65, 181-189. 45, 333-340. Smit, F. G. A. M. (1982). Classification of the Siphonaptera. In Synopsis and Kristan, D. M. (2002). Maternal and direct effects of the intestinal nematode Classification of Living Organisms, vol. 2 (ed. S. Parker), pp. 557-563. NY: Heligmosomoides polygyrus on offspring growth and susceptibility to McGraw-Hill. infection. J. Exp. Biol. 205, 3967-3977. Smith, H. J. and Archibal, R. M. (1969). Development of cross immunity Kumar, R. and Kumar, R. (1996). Cross-resistance to Hyalomma anatolicum in lambs by exposure to bovine gastrointestinal parasites. Can. Vet. J. 10, anatolicum ticks in rabbits immunized with midgut antigens of Hyalomma 286. dromedarii. Indian J. Anim. Sci. 66, 657-661. Studdert, V. P. and Arundel, J. H. (1988). Dermatitis of the pinnae of cats Leemans, I., Brown, D., Hooshmand-Rad, P., Kirvar, E. and Uggla, A. in Australia associated with the European rabbit flea (Spillopsyllus cuniculi). (1999). Infectivity and cross-immunity studies of Theileria lestoquardi and Vet. Record 123, 624-625. Theileria annulata in sheep and cattle: I. In vivo responses. Vet. Parasitol. Traub, R. (1980). The zoogeography and evolution of some fleas, lice and 82, 179-192. mammals. In Fleas. Proceedings of the International Conference on Fleas, Mans, B. J., Louw, A. I. and Neitz, A. W. H. (2002). Evolution of Ashton, England, June 1977 (ed. R. Traub and H. Starcke), pp. 93-172. hematophagy in ticks:common origins for blood coagulation and platelet Rotterdam, Netherlands: A. A. Balkema. aggregation inhibitors from soft ticks of the genus Ornithodoros. Mol. Biol. Uller, T. and Olson, M. (2003). Prenatal exposure to testosterone increases Evol. 19, 1695-1705. ectoparasite susceptibility in the common lizard (Lacerta vivipara). Proc. Marshall, A. (1981). The Ecology of Ectoparasitic . London: Academic Roy. Soc. Lond. B 270, 1867-1870. Press. Vatschenok, V. S. (1988). Fleas – Vectors of Pathogens Causing Diseases in McTier, T. L., George, J. E. and Bennet, S. N. (1981). Resistance and cross- Humans and Animals. Leningrad, USSR: Nauka Publ. House (in Russian). resistance of guinea pigs to Dermacentor andersoni Stiles, D. variabilis Vaughan, J. A., Jerse, A. E. and Azad, A. F. (1989). Rat leucocyte’s (Say), Amblyomma americanum (Linnaeus) and Ixodes capularis Say. J. response to the bites of rat fleas (Siphonaptera: Pulicidae). J. Med. Entomol. Parasitol. 67, 813-822. 26, 449-453. Metzger, M. E. and Rust, M. K. (1997). Effect of temperature on cat flea Warburg, A., Saraiva, E., Lanzaro, G. C., Titus, R. G. and Neva, F. (1994). (Siphonaptera: Pulicidae) development and overwintering. J. Med. Entomol. Saliva of Lutzomyia longipalpis sibling species differs in its composition 34, 173-178. and capacity to enhance leishmaniasis. Phil. Trans. R. Soc. Lond. B 345, Njau, B. C. and Nyindo, M. (1987). Detection of immune response in rabbits 223-230. infested with Rhipicephalus appendiculatus and Rhipicephalus evertsi Whiting, M. F., Carpenter, J. C., Wheeler, Q. D. and Wheeler, W. C. evertsi. Res. Vet. Sci. 43, 217-221. (1997). The Strepsiptera problem: phylogeny of the holometabolous Olsen, N. J. and Kovacs, W. J. (1996). Gonadal steroids and immunity. orders inferred from 18S and 28S ribosomal DNA sequences and Endocrine Rev. 17, 369-384. morphology. Syst. Biol. 46, 1-68. Poulin, R. (1996). Sexual inequalities in helminth infections: a cost of being Wikel, S. K. (ed) (1996). The Immunology of Host-Ectoparasitic Arthropod a male? Amer. Nat. 147, 287-295. Relationships. Wallingford, UK: CAB International. Rechav, Y. (1982). Dynamics of tick populations (Acari: Ixodidae) in the Wikel, S. K., Ramachandra, R. N. and Bergman, D. K. (1996). Arthropod Eastern Cape Province of South Africa. J. Med. Entomol. 19, 679-700. modulation of host immune responses. In Immunology of Rechav, Y. (1992). Naturally acquired resistance to ticks – a global veiew. Host–Ectoparasitic Arthropod Relationships (ed. S. K. Wikel), pp. 107-130. Insect Sci. Appl. 13, 495-504. Wallingford, UK: CAB International. Rechav, Y., Heller-Haupt, A. and Varma, M. G. R. (1989). Resistance and Wikel, S. K. and Alacron-Chaidez, F. J. (2001). Progress toward molecular cross-resistance in guinea-pigs and rabbits to immature stages of ixodid characterization of ectoparasite modulation of host immunity. Vet. ticks. Med. Vet. Entomol. 3, 333-336. Parasitol. 101, 275-287. Ribeiro, J. M. C. (1987). Role of saliva in blood feeding in arthropods. Ann. Willadsen, P. (2001). The molecular revolution in the development of Rev. Entomol. 32, 463-478. vaccines against ectoparasites. Vet. Parasitol. 101, 353-367. Ribeiro, J. M.C. (1995). Blood-feeding arthropods: live syringes or Willadsen, P. and Kemp, D. H. (1988). Vaccination with ‘concealed’ invertebrate pharmacologists? Infect. Agents Dis. 4, 143-152. antigens for tick control. Parasitol. Today 4, 96-198. Roehrig, J. T., Piesman, J., Hunt, A. R., Keen, M. G., Happ, C. M. and Willadsen, P., Eisemann, C. H. and Tellam, R. L. (1993). Concealed Johnson, B. J. B. (1992). The hamster immune-response to tick-transmitted antigens: expanding the range of immunological targets. Parasitol. Today Borrelia burgdorferi differs from the response to needle-inoculated, 9, 132-135. cultured organisms. J. Immunol. 149, 3648-3653. Zuk, M. and McKean, K. A. (1996). Sex differences in parasite infections: Rubaire-Akiki, C. M. and Mutinga, M. J. (1980). Immunological reactions patterns and processes. Int. J. Parasitol. 26, 1009-1024.